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Introduction: Dragendorff's reagent has low sensitivity and non‐specificity for some alkaloids. A new alkaloid derivatisation reagent has been developed and optimised by using a Box–Behnken design method. This new reagent is applicable for structurally diverse natural alkaloids, and is proposed as a universal alkaloid staining reagent. Objective: To establish an efficient and sensitive thin‐layer chromatography (TLC) identification method for Leonuri Herba and Leonuri Fructus to characterise their differences and similarities. Method: Three key components (bismuth subnitrate, potassium iodide and iodine) in the derivatisation reaction were re‐constructed and optimised using a response surface method. Different inorganic acids, essential additives of the staining reaction, were compared by a single‐factor experiment design. Results: This newly optimised reagent for alkaloids reported in this study, named the enhanced Dragendorff–Wagner's reagent, is composed of 0.82% bismuth subnitrate, 11.1% potassium iodide and 0.76% iodine in 38% phosphoric acid solution. Validation results indicate that the TLC spot of stachydrine stained with the enhanced Dragendorff–Wagner's reagent had a limit of detection of 2.0 μg, good intra‐ and inter‐plate, and intra‐ and intra‐day precisions with relative standard devition values less than 5.0%, and stability over 90 min. Conclusion: This enhanced Dragendorff–Wagner's reagent was applied for TLC identification of 16 reference alkaloids representing 11 structural skeletons, and two closely related herbs (Leonuri Herba and Leonuri Fructus). This newly enhanced Dragendorff–Wagner's reagent is a universal, effective, and sensitive staining reagent for alkaloids. A new derivatization reagent for TLC analysis of alkaloids, named enhanced Dragendorff‐Wagner's reagent, was re‐constructed and optimized. This enhanced Dragendorff‐Wagner's reagent was successfully applied for TLC identification of two closely related herbs (Leonuri Herba and Leonuri Fructus). This universal derivatization reagent was found to positive detection for 16 reference alkaloids representing 11 structural skeletons. [ABSTRACT FROM AUTHOR]

Introduction: Glucosinolates (1–5) are important secondary metabolites found in Isatis indigotica roots. Due to their high hydrophilic and ionic nature, purified glucosinolates often contain salt impurities and moisture. Accurate assessment of their purities is important for glucosinolates being utilised as chemical markers. Objective: To develop and validate quantitative proton (1H) nuclear magnetic resonance (qHNMR) methods for purity assessments of aliphatic and indole glucosinolates (1–5). Method: Several NMR parameters such as pulse program, relaxation time, and delay time were optimised. Three qHNMR methods were developed using gluconapin (3), neoglucobrassicin (4), and sinigrin (5) for method validation and with maleic acid as internal standard. Results: The quantification was based on the integrated area ratios of an olefinic proton (H‐4 for 1–3; H‐6 for 4; and H‐3 for 5) of the side chain from glucosinolates relative to the olefinic proton from the internal standard using deuterated water (D2O) as the solvent. The qHNMR methods were successfully applied for purity assessments of four aliphatic glucosinolates (1–3 and 5: progoitrin, epiprogoitrin, gluconapin, and sinigrin), and an indole glucosinolate (4: neoglucobrassicin). Conclusion: The purity of glucosinolates isolated from I. indigotica and commercial sinigrin was accurately assessed using the developed qHNMR method. The qHNMR provides a reliable and superior means to determine the purity of glucosinolates. Three qHNMR methods were developed and validated for purity assessment of aliphatic and indole glucosinolates using maleic acid as internal standard. The quantification was based on integrated area ratios of an olefinic proton of the side chain from glucosinolates relative to the olefinic proton from internal standard. The qHNMR method is a simple, rapid, irreplaceable, and accurate approach for purity assessments of glucosinolates. [ABSTRACT FROM AUTHOR]

Introduction TLC bioautography for tyrosinase inhibitors has made recent progress; however, an assay with a relative low consumption of enzyme and quantitative capability would greatly advance the efficacy of related TLC bioautographic assays. Objective An improved TLC bioautographic assay for detecting tyrosinase inhibitors was developed and validated in this study. Methods L-DOPA (better water-solubility than L-tyrosine) was used as the substrate instead of reported L-tyrosine. The effects of enzyme and substrate concentrations, reaction temperatures and times, and pH values of the reaction system as well as different plate types on the TLC bioautographic assay were optimised. The quantitative analysis was conducted by densitometric scanning of spot areas, and expressed as the relative tyrosinase inhibitory capacity (RTIC) using a positive control (kojic acid) equivalent. Results The limit of detection (LOD) of this assay was 1.0 ng for kojic acid. This assay has acceptable accuracy (101.73-102.90%), intra- and inter-day, and intra- and inter-plate precisions [relative standard deviation (RSD), less than 7.0%], and ruggedness (RSD, less than 3.5%). The consumption of enzyme (75 U/mL) is relatively low. Two tyrosinase inhibitory compounds including naringenin and 1- O-β- D-glucopyranosyl-4-allylbenzene have been isolated from Rhodiola sacra guided by this TLC bioautographic assay. Conclusion Our improved assay is a relatively low-cost, sensitive, and quantitative method compared to the reported TLC bioautographic assays. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Introduction The needle trap device (NTD) technique is a new microextraction method for sampling and preconcentration of volatile organic compounds (VOCs). Previous NTD studies predominantly focused on analysis of environmental volatile compounds in the gaseous and liquid phases. Little work has been done on its potential application in biological samples and no work has been reported on analysis of bioactive compounds in essential oils from herbal medicines. Objective The main purpose of the present study is to develop a NTD sampling method for profiling VOCs in biological samples using herbal medicines as a case study. Methodology A combined method of NTD sample preparation and gas chromatography-mass spectrometry was developed for qualitative analysis of VOCs in Viola tianschanica. A 22-gauge stainless steel, triple-bed needle packed with Tenax, Carbopack X and Carboxen 1000 sorbents was used for analysis of VOCs in the herb. Furthermore, different parameters affecting the extraction efficiency and capacity were studied. Results The peak capacity obtained by NTDs was 104, more efficient than those of the static headspace (46) and hydrodistillation (93). This NTD method shows potential to trap a wide range of VOCs including the lower and higher volatile components, while the static headspace and hydrodistillation only detects lower volatile components, and semi-volatile and higher volatile components, respectively. Conclusion The developed NTD sample preparation method is a more rapid, simpler, convenient, and sensitive extraction/desorption technique for analysis of VOCs in herbal medicines than the conventional methods such as static headspace and hydrodistillation. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Introduction – Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. Objective – To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. Methods – The new TLC bioautographic assay was based on reaction of lipase with β-naphthylmyristate and the subsequent formation of the purple dye between β-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. Results – The β-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07–105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64–4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8–4.9%). Conclusion – The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The mass spectrometric (MS) analysis of flavone di- C-glycosides has been a difficult task due to pure standards being unavailable commercially and to that the reported relative intensities of some diagnostic ions varied with MS instruments. In this study, five flavone di- C-glycoside standards from Viola yedoensis have been systematically studied by high performance liquid chromatography-electrospray ionization-tandem ion trap mass spectrometry (HPLC-ESI-IT-MSn) in the negative ion mode to analyze their fragmentation patterns. A new MS2 and MS3 hierarchical fragmentation for the identification of the sugar nature (hexoses or pentoses) at C-6 and C-8 is presented based on previously established rules of fragmentation. Here, for the first time, we report that the MS2 and MS3 structure-diagnostic fragments about the glycosylation types and positions are highly dependent on the configuration of the sugars at C-6 and C-8. The base peak (0,2X10,2X2− ion) in MS3 spectra of di- C-glycosides could be used as a diagnostic ion for flavone aglycones. These newly proposed fragmentation behaviors have been successfully applied to the characterization of flavone di- C-glycosides found in V. yedoensis. A total of 35 flavonoid glycosides, including 1 flavone mono- C-hexoside, 2 flavone 6,8-di- C-hexosides, 11 flavone 6,8-di- C-pentosides, 13 flavone 6,8- C-hexosyl- C-pentosides, 5 acetylated flavone C-glycosides and 3 flavonol O-glycosides, were identified or tentatively identified on the base of their UV profiles, MS and MSn ( n = 5) data, or by comparing with reference substances. Among these, the acetylated flavone C-glycosides were reported from V. yedoensis for the first time. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A combinative method using overpressured layer chromatography ( OPLC) and TLC bioautography against O2•− was developed to separate antioxidants from Glehnia littoralis roots. Through target-directed isolation by the TLC bioautographic method, seven compounds including five antioxidants were rapidly isolated by OPLC and identified as 1-linoloyl-3-palmitoylglycerol, facarindiol, panaxynol, isoimperatorin, β-sitosterol, scopoletin, and umbelliferone from G. littoralis roots. This OPLC method is a very suitable separation technique for light-sensitive polyacetylenes (panaxynol and facarindiol) with higher isolated yields compared to conventional open column chromatography. This is the first report on the separation of polyacetylenes by OPLC. [ABSTRACT FROM AUTHOR]

Ski8p is a WD-repeat protein with an essential role for the Ski complex assembly in an exosome-dependent 3′-to-5′ mRNA decay. In addition, Ski8p is involved in meiotic recombination by interacting with Spo11p protein. We have determined the crystal structure of Ski8p from Saccharomyces cerevisiae at 2.2 Å resolution. The structure reveals that Ski8p folds into a seven-bladed β propeller. Mapping sequence conservation and hydrophobicities of amino acids on the molecular surface of Ski8p reveals a prominent site on the top surface of the β propeller, which is most likely involved in mediating interactions of Ski8p with Ski3p and Spo11p. Mutagenesis combined with yeast two-hybrid and GST pull-down assays identified the top surface of the β propeller as being required for Ski8p binding to Ski3p and Spo11p. The functional implications for Ski8p function in both mRNA decay and meiotic recombination are discussed. [ABSTRACT FROM AUTHOR]